Institute of Biochemistry, Leipzig University, Johannisallee 21-23, Leipzig, Germany.
Institute for Organic Chemistry and Macromolecular Chemistry, Friedrich-Schiller-University Jena, Humboldtstr. 10, Jena, Germany.
Integr Biol (Camb). 2019 May 1;11(5):175-185. doi: 10.1093/intbio/zyz016.
Multiple cellular processes are affected by spatial constraints from the extracellular matrix and neighboring cells. In vitro experiments using defined micro-patterning allow for in-depth analysis and a better understanding of how these constraints impact cellular behavior and functioning. Herein we focused on the analysis of actin cytoskeleton dynamics as a major determinant of mechanotransduction mechanisms in cells. We seeded primary human umbilical vein endothelial cells onto stripe-like cell-adhesive micro-patterns with varying widths and then monitored and quantified the dynamic reorganization of actin stress fibers, including fiber velocities, orientation and density, within these live cells using the cell permeable F-actin marker SiR-actin. Although characteristic parameters describing the overall stress fiber architecture (average orientation and density) were nearly constant throughout the observation time interval of 60 min, we observed permanent transport and turnover of individual actin stress fibers. Stress fibers were more strongly oriented along stripe direction with decreasing stripe width, (5° on 20 μm patterns and 10° on 40 μm patterns), together with an overall narrowing of the distribution of fiber orientation. Fiber dynamics was characterized by a directed movement from the cell edges towards the cell center, where fiber dissolution frequently took place. By kymograph analysis, we found median fiber velocities in the range of 0.2 μm/min with a weak dependence on pattern width. Taken together, these data suggest that cell geometry determines actin fiber orientation, while it also affects actin fiber transport and turnover.
多种细胞过程受到细胞外基质和邻近细胞的空间限制的影响。使用定义明确的微图案进行的体外实验允许深入分析以及更好地了解这些约束如何影响细胞行为和功能。在此，我们专注于分析肌动蛋白细胞骨架动力学作为细胞中机械转导机制的主要决定因素。我们将原代人脐静脉内皮细胞接种到具有不同宽度的条纹状细胞粘附微图案上，然后使用细胞可渗透的 F-肌动蛋白标记物 SiR-actin 监测和量化这些活细胞内肌动蛋白应力纤维的动态重组，包括纤维速度、取向和密度。尽管描述整体应力纤维结构的特征参数（平均取向和密度）在 60 分钟的观察时间间隔内几乎保持不变，但我们观察到单个肌动蛋白应力纤维的永久性运输和周转。随着条纹宽度的减小（20 μm 图案上为 5°，40 μm 图案上为 10°），应力纤维更强烈地沿着条纹方向取向，同时纤维取向的分布也整体变窄。纤维动力学的特征是从细胞边缘向细胞中心的定向运动，纤维溶解经常发生在细胞中心。通过共聚焦分析，我们发现纤维速度的中位数在 0.2 μm/min 范围内，与图案宽度的依赖性较弱。总之，这些数据表明细胞几何形状决定了肌动蛋白纤维的取向，同时也影响了肌动蛋白纤维的运输和周转。
